Virtually all bacteria are host to viruses known as bacteriophages (hereinafter known as “phages”). These phages are species-specific, to the extent that a phage associated with the bacterium Enterococcus faecalis will be capable of infecting only Enterococcus faecalis, and a phage associated with Escherichia coli will infect only Escherichia coli. Viruses are non-cellular entities known as obligate parasites, which means they reproduce only within the cells of their specific hosts and they cannot replicate independently.
During lytic infection, phages will utilize the metabolic ‘machinery’ of the host and in the process multiply within the host cell to a point known as the “burst size”, at which point the host cell ruptures and releases the newly formed phages into the environment. The host cell is killed in the process. The newly formed phage particles may persist in the environment where they are ready to infect additional suitable host cells that they encounter. Because certain phages are excreted or egested by warm-blooded animals including humans, their presence in the environment may be interpreted as an indication of fecal contamination.
Because phages and enteric viruses are generally considered more resistant than bacteria to antagonistic environmental factors, phages may exhibit longer survival times than the bacteria currently used to determine the extent of fecal contamination in the environment. If that is the case, then it follows that testing the environment (water, food, etc.) for the presence of phages may yield more useful correlative evidence of viral contamination than would be the case when bacterial indicators are used.
Unfortunately, until now no simple, accurate method of testing for phages as environmental indicators has been available. In food and water testing, one of the most important indicator bacteria is Escherichia coli and various phages specific to it have been well described and documented. Since E. coli is by definition a “coliform” bacterium, the phages associated with it are known as “coliphages”. This bacterium and its viruses are well documented, the following discussion will be based on them. It should, however, be recognized that any microbial species and its phages might be used.
By minor changes in the nutrient formula and the use of different host bacteria, the methodology can be employed to detect any other bacteriophage specific to the host bacteria that can be propagated on a medium.
Currently, the approved methods for coliphage detection are detailed in Standard Methods for the Examination of Water and Wastewater, 20th edition as Method 9211 D., ISO Method 10705-2, ASTM Method 4201-96, and EPA Methods 1601/1602. These methods involve the use of agar based media which necessitates difficult and time-consuming temperature control procedures to maintain the integrity of samples and bacterial cultures. Because of the cumbersome, technique-specific nature of the Old Method(s), achievement of reproducible results is difficult for those laboratory technicians who lack experience with the methods or those who do not pay meticulous attention to detail, particularly as it relates to the temperature of the agar.
The approved enumerative methods are based upon the use of a semisolid matrix that functions to immobilize the host bacteria and the infecting phages. The medium must gel so that the bacteria grow within the gel-solid matrix, where they are attacked (infected) by phages present in the sample. When a bacterial host cell is infected with the virus, the virus reproduces within the host until the host cell is engorged with virus particles at which point it bursts and releases the viruses into the environment where they are free to infect new host cells. The consistency of the gel may affect the migration of phage particles in the medium. Those bacteria that are infected release many more phage particles which infect surrounding bacteria so that a clear zone of dead host cells appears in the “lawn” of dense living bacterial growth which covers most of the plate. This clear zone of necrosis is known as a viral “plaque” and the number of visible plaques is used to quantify the number of viruses originally present in the sample.
The use of coliphage qualitative and quantitative test methods are somewhat analogous to the testing for coliform bacteria in a given sample. Neither the coliphages nor the coliform bacteria are generally considered primary pathogens (disease-causing agents); rather, they are considered “indicators” of pathogenic contaminants that may be found in the same environment and their presence is generally assumed to indicate the potential presence of disease-causing microorganisms. Typical bacterial pathogens include species of genera such as Salmonella (typhoid and paratyphoid) or Vibrio (cholera). The absence of coliform bacteria is generally considered to reflect a likely absence of these and other bacterial pathogens because the indicator organisms are typically more numerous than pathogen bacteria in the environment and indicator and pathogenic bacteria generally exhibit similar survival times in the environment.
Because viral pathogens such as hepatitis A virus, the poliovirus group, noroviruses, and others tend to exhibit appreciable resilience in the environment and are characterized by extended survival times and in general to a high degree of resistance of antagonistic forces such as heat, freezing, and desiccation, for example, these viruses may be present even when the bacterial indicators are completely absent. Therefore, if a virus indicator such as a coliphage is used, there is an increased probability of capturing an actual virus contamination event when it occurs. Moreover, exclusive reliance upon bacterial indicators may result in the incorrect conclusion that pathogenic viruses are absent from the environmental sample.